Investigation of Chromosomal Abnormalities and Microdeletion/ Microduplication(s) in Fifty Iranian Patients with Multiple Congenital Anomalies

Objective Major birth defects are inborn structural or functional anomalies with long-term disability and adverse impacts on individuals, families, health-care systems, and societies. Approximately 20% of birth defects are due to chromosomal and genetic conditions. Inspired by the fact that neonatal deaths are caused by birth defects in about 20 and 10% of cases in Iran and worldwide respectively, we conducted the present study to unravel the role of chromosome abnormalities, including microdeletion/microduplication(s), in multiple congenital abnormalities in a number of Iranian patients. Materials and Methods In this descriptive cross-sectional study, 50 sporadic patients with Multiple Congenital Anomalies (MCA) were selected. The techniques employed included conventional karyotyping, fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and array comparative genomic hybridisation (array-CGH), according to the clinical diagnosis for each patient. Results Chromosomal abnormalities and microdeletion/microduplication(s) were observed in eight out of fifty patients (16%). The abnormalities proved to result from the imbalances in chromosomes 1, 3, 12, and 18 in four of the patients. However, the other four patients were diagnosed to suffer from the known microdeletions of 22q11.21, 16p13.3, 5q35.3, and 7q11.23. Conclusion In the present study, we report a patient with 46,XY, der(18)[12]/46,XY, der(18), +mar[8] dn presented with MCA associated with hypogammaglobulinemia. Given the patient’s seemingly rare and highly complex chromosomal abnormality and the lack of any concise mechanism presented in the literature to justify the case, we hereby propose a novel mechanism for the formation of both derivative and ring chromosome 18. In addition, we introduce a new 12q abnormality and a novel association of an Xp22.33 duplication with 1q43q44 deletion syndrome. The phenotype analysis of the patients with chromosome abnormality would be beneficial for further phenotype-genotype correlation studies.


Introduction
Major birth defects are considered to be inborn structural or functional abnormalities which could be diagnosed prenatally, at birth, or later in infancy, or even in adulthood. Their consequence will be a long-term disability with major adverse effects on individuals, families, health-care systems, and societies (1). Approximately 20% and more than 10% of all neonatal deaths in Iran and worldwide (2) are caused by birth defects, respectively. The incidence of major birth defects in neonatals, resulting from genetic or partially genetic factors, is about 7.9 million per year (3). A fifth (20.2%) of the cases with birth defects are attributed to a known etiology comprising of chromosomal (15.8%) and genetic (3.8%) conditions. Teratogens are the cause of 0.8% of cases, 0.3% of them are caused by twinning, and four-fifths (79.8%) are categorized to be of unknown etiology (1). Nearly, 20 to 30% of the infants suffering from birth defects have multiple congenital anomalies (MCA) involving different organs. In cases where two or more major congenital abnormalities occur in several organs and the defects do not represent a sequence or a complex series, the case is classified as MCA (4).
Genetic factors are regarded as one of the most prominent etiologies of MCA (1). Also, chromosomal abnormalities have been known to be one of the leading causes of intellectual disability (ID) as well as congenital malformations. A large number of chromosomal defects can be detected immediately through high-resolution karyotyping technique. But, the resolution of the cytogenetic analysis is restricted to around 5 to 10 Mb. Among molecular cytogenetic techniques, fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) are two of the targeted tests used to detect submicroscopic chromosome abnormalities. Genomewide molecular cytogenetic tests like array comparative genomic hybridisation (array-CGH) can identify a variety of copy number variants (CNVs) associated with MCA. Array-CGH, as a powerful test, is applied to investigate individuals with MCA, ID, and autism spectrum disorders (5). In the current study, we aimed at identifying the chromosomal abnormalities and microdeletion/microduplication(s) in 50 Iranian patients with MCA. The patients manifested diverse phenotypes including ID/developmental delay (DD), and at least one major congenital anomaly in another organ. Existence of additional minor dysmorphic features was also considered among the factors representing chromosomal abnormalities which led to an increase in the detection rate.

Selection of patients
In this descriptive cross-sectional study conducted during the three past years from April 2015 to May 2018, clinical evaluation was performed for 50 selected sporadic patients suffering from MCA preferably born to unrelated parents and referred to us by experienced clinical specialists for genetic investigation from all over the country. All of the patients had ID/DD with at least one major anomaly and additional minor dysmorphic features. All of the steps taken for testing and procedures were fully explained for all of the probands' parents, and signed informed consent forms from all of the participants were obtained for publishing any information or accompanying photographs. This study was approved by the Ethics Committee of the University of Social Welfare and Rehabilitation Sciences (IR.USWR.REC.1394.186).

Conventional karyotyping
A conventional cytogenetic study was carried out on peripheral blood lymphocytes using GTG high-resolution banding technique according to standard protocols for all patients (6). Twenty GTG banded metaphases were examined through the complete analysis of each individual sample. Chromosome analysis was performed according to ISCN 2016 (7).

Fluorescence in situ hybridization
Metaphase FISH was done only for patient 9 using the centromeric probe of chromosome 18 (Kreatech/ Leica Biosystems Buffalo Grove, IL, USA, http://www. leicabiosystems.com) according to Kreatech protocol (8). Two hundred cells were examined to characterize the ring chromosome origin defined in 37% of cells by highresolution GTG banding.

Multiplex ligation-dependent probe amplification
MLPA technique was done using P245 Microdeletion Syndromes-1 kit (MRC-Holland, Amsterdam, the Netherlands) for the patients suspected of microdeletion/ microduplication syndromes. MLPA protocol was performed based on the instructions provided by the manufacturer, MRC Holland, and the data were analyzed utilizing capillary electrophoresis in the 3130XL DNA Analyzer, Coffalyser. Net software (MRC-Holland, Amsterdam, the Netherlands) and Gene Marker software version 2.7.0 (Softgenetics, State College, PA, USA). Abnormal results obtained with MLPA assay were repeated and if different, further investigations were carried out with array-CGH. MLPA studies were done for the patients' parents with abnormal results in order to determine the causal role (de novo or inherited) of microdeletions.

Array comparative genomic hybridization
The analysis of oligonucleotide array-CGH for Genomic DNA was carried out. To do so, the BlueGnome CytoChip ISCA 8×60 K v2.0 wholegenome oligo array was utilized for patients 1, 5, 9, 13, and 41. This array included intragenic and intergenic probe spacings of about 48 kb and 70 kb, respectively and high probe density in 500 clinically important regions. CytoSNP-850K v1.1 BeadChip overall effective resolution of about 18 Kb was used for patient 27. The tests were performed based on the manufacturer's protocol. INNOPSYS 910 laser scanner was utilized for scanning according to the recommended protocol of the manufacturer. Image analysis and base calling were carried out employing the BlueFuse Multi-version 3 analysis software for oligo array and Multi-version 4.4 analysis software for SNP array. The analysis of the samples was carried out two times against two non-identical controls and representation of minimum three clones on the platform in both of the experiments was considered to approve the imbalances. To evaluate the called CNVs, we utilized the standards and guidelines of American College of Medical Genetics and Genomics to interpret the postnatal constitutional CNVs (5). All chromosome coordinates are on the basis of GRCh37. p13/hg19.

Results
Based on the selection criteria, the patients had MCA, i.e., having ID/DD and at least one major congenital anomaly in another organ plus additional minor facial dysmorphic features. The clinical characteristic features of all patients with MCA are summarized in Table 1. The participants included 25 male and 25 female patients. Their ages ranged from 7 months to 21 years with a median age of 3.25 years and an average of about 5.1 years (Table 1).
In patient 9, a 31-month-old boy from consanguineous parents (half first cousins) and born to a 27-year-old G1P1L1A0 (G: gravid, P: parity, L: live birth, A: abortus) mother, the clinical features were global DD, PDA, PFO, optic nerve hypoplasia, hypogammaglobulinemia, facial dysmorphic features, microcephaly, and mild ventriculomegaly in frontal horns in the axial spiral CT-scan of the brain without contrast, and benign extra-axial hydrocephalus and atrophic changes in the brain magnetic resonance imaging (MRI). The karyotype analysis by highresolution GTG banding showed 46,XY, der (18) [12]/46,XY, der(18), +mar [8] dn (Fig.1A1, 2). Since in both cell lines of the patient the short arm of one chromosome 18 (18p) was abnormal and one cell line had an additional ring chromosome, one hundred cells were screened to obtain the percentage of the cells with marker ring chromosome. The karyotype analyses of the proband's parents were normal. Metaphase FISH using the centromeric probe for chromosome 18 indicated three hybridization signals for chromosome 18 in 37% of the cells scored which was consistent with mosaic trisomy 18, nuc ish(D18Z1×3) [37]/(D18Z1×2) [63] dn (Fig.1A3, 4). Further investigations using array-CGH demonstrated the exact breakpoints of the derivative chromosome 18 and its deletion and duplication (Fig.1A5).      The clinical findings for patient 9 were compared with the previously reported patients in the literature and the ECARUCA database (http://www.ecaruca.net) (9) in Table 3.

Chromosomal Abnormalities in Congenital Anomalies
The cytogenetic and molecular cytogenetic results of 8 patients were reported in detail in Table 4.
Mohammadzadeh et al.  Patient 17, an 8.5-month-old boy born to nonconsanguineous parents with DD, infantile spasms and myoclonic jerks, abnormal EEG, PDA, ASD, Facial dysmorphic features, Hypotonia, and Corpus callosum hypoplasia (Table 1), showed an additional segment of 12q15-qter origin on 4q33 in karyotype analysis. The patient father's karyotype was normal, but his mother's karyotype showed a reciprocal balanced translocation between long arms of chromosomes 4 and 12 46,XX,t(4;12)(q33;q15). The clinical features of patient 17 were compared with those of the previously reported patients who had overlapping genotypes with patient 17. The information was obtained from the ECARUCA (9) and DECIPHER (16) databases (Table 5).
The karyotype of patient 27, an 18-year-old boy from non-consanguineous parents and born to a 31-year-old G7P6L6A1 mother at term, had been reported as a normal male in his medical documents. The result of further investigation using P245 Microdeletion Syndromes-1 kit was negative for the microdeletion/microduplication syndromes. The clinical features were ID, tetralogy of fallot (TOF), microcephaly, severe scoliosis, short stature, speech delay, and facial dysmorphic features (Table 1). In the conventional cytogenetic investigation repeated, a suspicion for terminal 1q deletion was observed. Array-CGH was performed and reported two inherited CNVs: A 7.2 Mb deletion of the terminal end of 1q43q44 and a 541 Kb duplication of Xp22.33. The array-CGH analyses performed for both of the parents were normal.
Patient 1 was a 14-month-old girl from healthy and young non-consanguineous parents born through normal vaginal delivery to a G1P1L1A0 mother at term. The clinical characteristics were ID, intrauterine growth restriction (IUGR), hypotonia, hypothyroidism, supravalvular aortic stenosis, supravalvular pulmonary stenosis, left visual defect, attention deficit/hyperactivity disorder, strabismus, long philtrum, and thick lips. Conventional cytogenetic analysis was normal. MLPA screening demonstrated a deletion of 7q11.23 consistent with Williams-Beuren syndrome. Further assays using array-CGH confirmed about 1.37 Mb deletion including 22 OMIM genes. Array-CGH analyses of the parents were normal. Patient 5 was a 5.5-year-old girl, born through normal vaginal delivery to healthy and young non-consanguineous parents following a full-term pregnancy. Her mother was G2P1L1A1. She had suffered constipation for the first six months after birth. At the age of 14 months, the renal cortical scan revealed lesser activity in the lower pole of the left kidney because of pyelonephritis. During voiding cystourethrogram, severe urinary tract reflux was present in the left kidney. She also had a large ASD, ID, seizure attacks, autistic behavior, macrocephaly, dolicocephaly, frontal bossing, fish-shaped mouth, sparse hair, and joint laxity. She can say up to 5 words. The karyotype analysis was normal. MLPA assay revealed a deletion of 5q35.3 consistent with Sotos syndrome. Further investigations using array-CGH confirmed 1.86 Mb deletion including 25 OMIM genes. Array-CGH analyses of the parents were normal.
Patient 13 was a 3-year-old boy who was the product of the first pregnancy and cesarean delivery of nonconsanguineous parents. The clinical features were widow's pick hair, down-slanting palpebral fissures, long eyelashes, deep set eyes, malformed, posteriorly rotated and low set ears, upward nose and nares, long philtrum, small mouth, high arched palate, high pitched cry, wheezing respiratory sounds, cardiac surgery scar, simian crease, 5 th toe clinodactyly, umbilical hernia, sacral mongolian spot, overriding of 1 st toe with 2 nd toe, right lower limb paresis, joint contractures, right sided tippy-toed walking and bilateral undescended testes. Axial spiral CT-scan of the brain without contrast media showed bilateral pachygyria at frontotemporoparietal lobes due to abnormal neuronal migration. The karyotype analysis was normal. MLPA assay detected a deletion of 22q11.21 consistent with DiGeorge syndrome. Further investigations using array-CGH confirmed 2.86 Mb deletion including 41 OMIM genes, arr 22q11.21 (18,706,023-21,561,492) x1 dn. Array-CGH analyses of the parents were normal.
Patient 35 was a 7-month-old girl born through cesarean section to a healthy consanguineous (first cousin) couple. Her mother was G1P1L1A0. She had bilateral cataracts, hypertelorism, left ptosis, frontal bossing, low set ears, long philtrum, broad nasal bridge, micrognathia, a borderline lactic acid and normal creatinine in urine, ascending aorta dilatation, peripheral pulmonary stenosis, and small ASD. Conventional cytogenetic analysis showed a normal female karyotype. The MLPA technique indicated a deletion of 16p13.3 consistent with Rubinstein-Taybi syndrome. The sample assay was carried out twice. The parents' karyotype analyses and MLPA assays were normal.

Discussion
Based on several studies, the diagnostic yields of conventional cytogenetic studies in patients with MCA, global DD, and autism spectrum disorders, barring the known trisomy syndromes, have been reported to be about 3% (20). In this study, we identified chromosomal abnormalities, microdeletions, and microduplications for 8 patients (16%) using conventional karyotyping technique [3 patients (6%)], MLPA method [4 patients (8%)], and array-CGH [1 patient (2%)]. In addition to the sample size limitations, the adoption of inflexible criteria for selecting patients and the design type of the study could have contributed to the raising the diagnostic rate of this study.
Well-defined microdeletion syndromes were the most common cause of MCA in this study, consistent with the results of previously reported studies (21). The clinical features of four patients 1, 5, 13, and 35 are congruent with known microdeletion syndromes namely Williams-Beuren syndrome, Sotos syndrome, DiGeorge syndrome, and Rubinstein-Taybi syndrome, respectively. Consequently, they are not discussed in detail.
According to a hypothesis, two critical regions are within the long arm of chromosome 18 (18q), including 18q12.1-q21.2 and 18q22.3-qter. Duplication occurrence is essential in both proximal and distal regions in order to cause the typical clinical features of a trisomy 18 to be manifested (12). In patient 9, the former duplication is considered as a mosaic trisomy and the latter as a full trisomy.
As a chromosomal abnormality, partial deletion of 18p is more common than partial duplication of 18q (22). Mostly, a balanced translocation or inversion carried by one parent is the leading cause of partial trisomy of 18q (15). The real mechanism of mosaic ring 18 formation in patient 9 is unknown. Although his parents' karyotypes were normal, there was a recombinant pattern of pericentric inversion and an extra ring in the patient's karyotype. The possible mechanism for the formation of both derivative and ring chromosome 18 could, most probably, have initiated with an inverted chromosome 18 (p11.21-q21.31), created during meiosis in the gamete of one parent (23). Postzygotic mitosis resulted in two partially q duplicated and partially p deleted recombinants (22). The genetic size of 18p is almost 16 Mb with a common breakpoint cluster located in the pericentromeric region with a length of 4 Mb. The partial deletion of 18p in this patient was inside the breakpoint cluster spanning 13.3 Mb. It should be noted that the 18q21.3 segment is one of the common fragile sites of chromosome 18 (24). One of the recombinants had seemed to have two breakpoints in 18q21.31 and 18p11.21 bands and the sticky ends of the segment between them fused and formed the ring chromosome 18. The broken segments were lost and some of the rings created were deleted in the next mitoses. The formation of ring chromosome 18 may be a novel escape mechanism in this patient with a pentasomy of 18q21.31qter segment (25).
Most clinical phenotypes of patient 9 overlapped with the clinical manifestations of partial 18p deletion, partial 18q duplication, and mosaic ring chromosome 18 syndromes. The phenotypic variations could be explained based on the locations and the lengths of the duplicated or deleted segments with a mosaic triplicated segment between them considering the co-occurrence of different types of chromosome 18 abnormalities in this patient.
Litzman et al. (13) reported an agammaglobulinemic 14-year-old girl with a mosaic ring chromosome 18. Her karyotype turned out to be: 45, XX,-18 [5]/46, XX, dic r(18) [6]/46, XX, r (18)[89]. IgA deficiency/absence with normal ranges of IgG and IgM has been previously reported in association with 18p deletion (11). To the best of our knowledge, the proband is the first patient to be reported with partial monosomy of 18p, partial 18q duplication, and an extra mosaic ring chromosome 18 associated with hypogammaglobulinemia.
Based on the information obtained, no patient has been previously reported with partial duplication of 12q with the breakpoint at 12q15 to qter. However, although chromosome 4q deletions occur rarely, 4q33-qter deletions have been reported several times (17). It seems that some clinical features of patient 17 like hypoplasia of corpus callosum, hypotonia, and high-arched palate are also observed in the cases with partial 12q duplication. In contrast, some of the other physical characteristics such as heart defects, flat midface, camptodactyly, and depressed nasal bridge are consistent with phenotypic features of terminal deletion of 4q33-qter. However, a number of major anomalies in patient 17 including bilateral hearing loss, imperforated anus, umbilical hernia, and small kidneys have not been reported in the patients with partial 12q duplication or partial 4q deletion. The large size of 12q15-qter duplication could be the cause of these extra phenotypes. Homo sapiens HAND2 gene located on 4q34.1 has been known as the responsible gene whose proteins play a crucial role in developing ventricular chambers, cardiac morphogenesis, forming right ventricle and aortic arch arteries as well as developing limb and branchial arch. The haploinsufficiency of this gene in 4q deletion may justify inborn heart anomalies (i.e., VSD and ASD), growth retardation, ID, digital anomaly (i.e., clinodactyly and oligodactyly), and craniofacial dysmorphism including a cleft palate (26).
Approximately 11.4 Mb deletion of the distal segment of 3p containing 46 OMIM genes has led to severe and different phenotypes in patient 41.
The distal deletion of 3p is a rare contiguous gene syndrome with the variable spectrum of major anomalies such as developmental delay, growth retardation, autism, hearing loss, renal anomalies, heart defects, and craniofacial dysmorphic presentations. Given common facial dysmorphic features and ID among patients with intragenic sequence variants and microdeletions affecting SETD5, it has been suggested that this gene plays a vital role in ID and also has a significant contribution to the core phenotype of 3p deletion syndrome (27).
It seems that ID severity in patient 41 is due to the haploinsufficiency association of SETD5 and BRPF1 genes. CHL1 gene deletion associated with mild ID and learning difficulties (28) may also contribute to this severity. Furthermore, some clinical manifestations specially ptosis and short stature may occur because of BRPF1 gene deletion (29).
The autistic behavior of patient 41 may be because of CNTN4 (contactin 4), previously introduced as a strong candidate gene associated with autism spectrum disorders (30). Two genes namely SLC6A1 and SLC6A11 encoding GABA transporter proteins have been proposed as possible candidates for seizures/EEG abnormalities, ataxia, and ID (31).
1q43q44 deletion in patient 27 with the size of 7.2 Mb included 126 HGNC and 19 OMIM genes. Of these genes, AKT3 gene haploinsufficiency has been linked with microcephaly in previously reported cases with 1q43q44 deletion syndrome (32). However, because of the presentation of microcephaly in the patients with intact AKT3 gene, the other candidate genes such as NLRP3, HNRNPU, SMYD3, and KIF26B have been suggested to cause microcephaly (33). Nonetheless, AKT3 deletion may explain the severity of microcephaly in patient 27.
Corpus callosum agenesis/hypoplasia and epilepsy are mostly associated with deletions of ZBTB18 and HNRNPU genes in the patients with 1q43q44 deletion syndrome, respectively (34). However, none of these symptoms is demonstrated in the present case despite ZBTB18 and HNRNPU disruptions. Recent studies have suggested that modifier genes or two-hit hypothesis may explain variable expressions among some patients with identical microdeletions. Another CNV is a disruptive singlebase-pair mutation in a related gene or an environmental event which influences the phenotype. These are all the examples of the second hit (35).
Patient 27 also had a duplication of about 541 Kb in Xp22.33 containing PLCXD1, GTPBP6 and PPP2R3B genes as well as exons 1-3 and part of exon 4 of SHOX gene. PPP2R3B is one of the four major Ser/Thr phosphatases and negatively affects cell growth and division. PPP2R3B performs a regulatory control over the starting of DNA replication and its overexpression results in G1 phase cell cycle arrest (36).
There has been a variable impact on stature due to duplications of the region PAR1 containing the SHOX gene. Although some studies point to a correlation between SHOX gene deletions and short stature and Leri-Weill dyschondrosteosis, others have found some patients with the same clinical features who had partial or full duplications of SHOX gene. Bunyan et al. (37) reported a duplication of the exons 1-3 of the SHOX gene in a patient who had only short stature with no other clinical findings inherited from the proband's affected mother. Co-overexpression of PPP2R3B and SHOX genes seems to have a synergistic effect on our proband's short stature severity.

Conclusion
In this study, 50 patients with MCA were studied in terms of chromosomal abnormalities and microdeletion/ microduplication syndromes. A patient was introduced with 46,XY, der(18) [12]/46,XY, der(18), +mar [8] dn as a novel case of MCA associated with hypogammaglobulinemia. Afterwards, given the highly complex and rare nature of the patient's chromosomal abnormality, a novel mechanism was proposed to explicate the formation of both derivative and ring chromosome 18. Furthermore, a new 12q abnormality was found which had not been reported previously. In addition, a novel association of an Xp22.33 duplication with 1q43q44 deletion syndrome was demonstrated. The phenotypic analysis of the introduced patients can be useful for further phenotype-genotype correlation studies.